Team:Toulouse/Results
Results
Content
Attract
Tests on varroas
In the US patent US 8647615 B1, the concentration of butyric acid that attracts varroa mites is 4 % (V/V). In the final description it is specified that an efficient amount of attractant may at least be 0.00001 %.
In order to verify the results presented in this patent, we designed an attraction test on varroas. Champollion University in Albi welcomed us in their lab to perform this test, but there were not a lot of varroas available so we chose to make only one test in order to have a significant result. Hence, we used the mentioned 4 % butyric acid concentration, like the one first mentioned in the patent.
|
|
Figure 1: Butyric acid test pie chart and statistical test
This test demonstrates that a solution of 4 % butyric acid attracts varraos. However, in the Cytotoxicity part of the results, we also show that this 4% concentration is totally lethal for bacteria, so our goal is to produce a concentration of butyric acid of at least 0,00001 % (as described in the patent).
For further experimentation, it would be interesting to do another attraction test with the right concentration, i.e. the one we could produce.
Cloning butyrate genes
We ordered synthetic genes designed by us to get the full regulated pathway, so the ccr gene was already placed behind lacI, ready for circadian circle regulation. We therefore cloned the ccr gene with all genes necessary for butyrate production, in order to have the construction below:
Figure 2: assembly of the synthetic pathway leading to butyrate production (5220 Kb). The first arrow represents the promoter, the others represent genes, the green circle is for the RBS and red circle is for terminator. Purple genes originated from Streptomyces collinus, blue genes form Clostridium acetobutylicum and yellow genes from Escherichia coli.
This construction (BBa_K1587004), inserted in pSB1C3 is in the biobrick format and was then verified by an EcoRI and PstI digestion, leading to the release of the inserted fragment. This confirmed the right insertion of the fragment.
Figure 3: Gel electrophoresis of the digestion of the (BBa_K1587004) verification of the inserted fragment encoding the pathway to produce butyrate.
The size of the first DNA fragment matches 5192 pb (butyrate construction) and the size of the second one (2070 pb) matches linearized pSB1C3.
Test of butyrate production
In order to test butyrate production, we cultivated ApiColi under micro-aerobic conditions. After having haversted the supernatant of the culture, we filtrated it prior to do NMR analysis.
We tested our genetic construction in the E. coli strain BW25113 but were unable to detect butyrate production. As a mean to check if the pathway introduced was actually operating, we also searched for significant difference in other products found in the culture medium: acetate and ethanol. We also tested the butyrate production with a strain deleted for phosphate acetyltransferase, as explained in “Metabolic Engineering of Escherichia coli for Production of Butyric Acid” [1] and in the figure below.
Figure 4: Exogenous acetate system. Deleted genes are coloured red. Reference [1]
Even if other enzymes for butyrate production were used, as explained in Attract part, this metabolic pathway figure can be helpful to understand which genes would need to be deleted for butyrate production.
Indeed, in the publication, three others genes were also deleted to produce butyrate. However, the lack of time did not allow us to perform these experiments. Only pta deletion could be achieved.
Figure 5: Test of butyrate production in E. coli strain deleted for pta gene. Culture in micro-aerobic condition in 10 mL falcon, result obtained after 28.5 hours culture.
Unfortunately we could not detect butyric acid with our NMR analysis, but we see differences for others fermentation products, showing up that something has happened in our constructed strain. Our bacteria produced less formate and a little more acetate but the biggest difference is visible for ethanol which is lower than in the control strain. It is be possible that Acetyl-CoA is transformed in Acetoactyl-CoA thanks to enzyme we added. Then Acetyl-CoA would be less available to be transformed into ethanol. We did not detect these intermediate metabolites because they are not released into the medium. In any case, our genetic construction modified the fermentative balance.
In a forthcoming experiment it would be useful to measure intracellular metabolites to see if intermediate products are actually synthesized. Moreover, it would be very interesting to test our genetic construction with a strain deleted for all genes indicated in figure 4.
Eradicate
Tests on varroas
In order to determinate what target quantities of formic acid need to be reached during the killing phase, we tested different concentrations of formic acid on varroas as explained in the Protocol part.
Figure 6: Mortality of varroas as a function of time for different formic acid concentrations
Figure 7: Histogram representing mortality of varroas after 2 hours and after 7 hours
Figure 6 presents a dose-dependency of formic acid on varroa mortality. At 10mM formic acid, all varroas died before three hours but as we explain in Protocol part varroas also stop moving at lesser concentrations. Figure 7 shows that even with 50µM of formate, around 30 % varroas died after 7 hours. As our first goal is to respect bees, we wanted to produce as little as possible of formic acid. That is why we set our production goal around 50µmol.L-1 during the night (7 hours).
Test of formate production
For formate production we designed genes coding for pyruvate formate lyase and the activation protein. The assembly was cloned in pUC57 plasmid (from the gene synthesis company) so we could directly test formate production without any cloning step. We then made a BioBrick out of this : BBa_K1587007.
Figure 8: Substrate and products concentration for formate production under micro-aerobic conditions
Figure 9: Summary of formate production tests after 3 days cultivation under micro-aerobic conditions
Figure 8 shows that the only difference between ApiColi and the control was the formate production, so we plotted the specific histogram for formate. Figure 9 indicates that formate production increased significantly by 10%.
As mentioned earlier, our goal was to produce 50µM of formic acid in 7 hours. To reach this quantity, we needed to produce 77mM of formate. We obtained a formate production of 25mM in 36 hours (Figure 8), around 5mM in 7 hours. Therefore our production was at the same level than the targeted production.
Thanks to our device considerations and all the results summarized below, we show that it will be possible to reach our target production with slight optimizations of the metabolic pathway regulation.
ApiColi containment and culture
Growth tests in TPX bag
The second step was to know if bacteria can grow inside a small bag of TPX®. Thus, the strain E. coli BW 25113 has been inoculated in a small bag and further added inside a tube and incubated at 37 °C. The monitoring of OD at 600 nm has been performed over 10 days.
Figure 10: Growth test of E. coli BW 25113 inside a small bag of TPX® (Tubes n°1 and n°2).
Both tubes contain a small bag of TPX® in which bacteria are growing (t = 17 hours, 37 °C).
Figure 11: Growth test of E. coli BW 25113 in a culture tube (Tube 3).
The culture tube, contains bacteria which are growing (t = 17 hours, 37 °C, 130 rpm) in parallel of biological sample tubes shown above. It represents the control: E. coli BW 25113 are growing under aerobic condition with agitation.
The graph below shows the monitoring of OD at 600 nm. The strain E. coli BW 25113 is growing over 10 days (Tube n°1') in the TPX® bag .
Figure 12: Growth test in TPX® bag by monitoring OD at 600 nm over 7 days (tubes n°1 and 2) over 10 days (tube n°1').
The picture below represents the strain of E. coli BW25113, previously identified as tubes n°1', n°1, n°2 and n°3 after 10 days of culture for tube 1' and 7 days for tubes 1, 2 and 3.
Figure 13: Colonies of E. coli BW 25113 on Petri dishes after an overnight incubation at 37°C to check survivability.
Bacteria are still alive after 10 days or 7 days (depending on the tube) while growing in TPX® bags. In all the culture tubes, the number of cells is about 4.106. Thus, the strain can survive over 10 days in the TPX® bag.
Gas diffusion tests
In order to verify if our test (described in the Protocol section) works, we performed a series of control reactions using butyric acid solutions in a Falcon and we quantified the gases.
We did not detect any evaporation of butyric acid by NMR and postulated that the solution we used to solubilize gases may not be sufficiently alkaline. We then performed a test with a TPX® bag containing butyric acid into a solution of sodium bicarbonate. The control is an injection of a 4% (V/V) solution of butyric acid in water.
Figure 14: NMR Spectrum of butyric acid liquid control in red and butyric acid liquid which passed through TPX bag in blue. * Blue curve is zoomed 1340 times more than red curve. Each condition was tested in two replicates.
Table 15: Concentrations of butyric acid calculated from the NMR spectrum
From these results, we can conclude that TPX® allows butyric acid
to pass outside the bag. We detect only a small quantity but
an optimization of the device could be made with a plastic containing
bigger pores in order to let more butyric acid go out.
For formic acid we were able to detect it
in gas, probably because its pKa is lower than butyric acid one.
Figure 16: NMR spectrum of formic acid gas control in red and formic acid gas which passed through TPX bag in blue. An internal standard present in both curves allowed us to standardize both curves. curves. Each condition was tested in two replicates.
Table 2: Concentrations of formic acid corresponding to NMR spectrum
According to these results, TPX® allows 56% of formic acid to pass outside the bag in gas phase. We show that formic acid can go through TPX plastic. Using a more porous plastic, we propose that this percentage could even further increase.
Safety tests
The bacteria impermeability of TPX®, has been tested through inoculation of the strain
E. coli BW 25113 in M9 defined medium. To summarize, the strain (in M9 medium) has been inoculated inside the small bag of TPX®. Then, the inoculated bag was immersed in a glass measuring cylinder containing M9 medium. OD600 nm served to monitor and growth in the external medium.
Figure 17: Measuring cylinders used for the safety test of the TPX® polymer.
The first cylinder, on the right contains the small TPX® bag with E. coli BW 25113 after 27 hours of growth at 37 °C. On the right, the negative control cylinder contains a small bag of TPX® without bacteria and immersed in M9 medium after 27 hours of growth at 37 °C.
Over this time, no bacteria went out of the bag, so the sterility has been conserved over 27 hours.
Growth tests
In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
and formic acid during at least ten days in order to be practical for beekeeper.
So we faced some biological questions:
- Could bacteria live during ten days in micro-aerobic condition?
- Which carbon source could we have to produce continuously acids?
- Would acids be toxic for E.coli?
Characteristics of E.coli growth
In order to know better the E.coli strain we would use for our project, we made a culture in aerobic and micro-aerobic conditions. We sampled OD and supernatant to see what happened in it.
Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes covered with a membrane into the plug which let the oxygen pass through without opening the falcon. They were incubated at 37 °C without agitation to best correspond to our real condition.
Aerobic condition is obtained with a classical Erlenmeyer incubated at 37 °C with agitation.
For the medium, we use a minimal medium M9 because we want to follow acids production by NMR. And we chose a standard glucose concentration, 15mM.
Biomass, substrate and products
In order to plot biomass concentration it is necessary to convert the OD measured.
This equation was used:
$$ X=OD_{600nm}\times 0,4325 $$
Where X is the cell concentration (g.L-1)
For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
Then, we calculated concentration with this equation:
$$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$
- [A] = concentration of molecule in our solution in mM
- AreaTSP = 1
- [TSP] = 1.075 mM
concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for quantification - TSP proton number = 9
- DF = Dilution Factor = 1.25
Thanks to these calculations we were able to plot biomass, substrate and products depending on time.
Figure 18: Results of aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Erlenmeyer at 37 °C
Figure 19: Results of micro-aerobic culture. Culture of E. coli BW25113 in M9 medium with [glucose] = 15 mM, in Falcon at 37 °C
Glucose is consumed approximately at the same rate for both conditions but it is not use for the same thing at all. In aerobic condition biomass reaches 3 g/L whereas in micro-aerobic condition there is six times less biomass. To the contrary, there are far less products in aerobic conditions, and bacteria consume them when there is not glucose anymore, than in micro-aerobic condition.
For our objective to produce acids in a microporous bag, it is a really interesting results to have naturally bacteria which have slow growth and fermentation products.
We can convert formate
concentration into formic
acid to know how much more
we will have to produce to
kill varroa. Indeed, the bacteria
produce a base but it is the acid that
interests us.
The formula below
is used:
$$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$
- pH: medium used is buffered with a low concentration in acid. pH = 7.
- pKa: 3.7 for formic acid and 4.81 for butyric acid
- Cb: base concentration
- Ca: acid concentration
As it is said in the Eradicate> part, our goal is to produce 50 µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds to 77,7 mM of formate.
At the maximum the bacteria produces 32mmol/L of formate. It is necessary to add genes involved in formate production to regulate production and increase it by 240%. For a perfect regulation it would be necessary to delete pflB in E.coli genome to avoid formate production by day.
Bacteria survival
As it is explained here we plated bacteria on Petri dish to know if they were alive or not because OD measure cannot discriminate alive bacteria from dead. This test show us that wild type bacteria can easily survive during at least 15 days. So if we bring them a carbon source during this period they should survive even better.
Figure 20: Bacteria survival results from culture test with BW25113 on M9 with 15mM of glucose during 15 days to mime real survival condition.
Choice of carbon source to produce acids during 10 days
Characteristics of Biosilta kit
En Presso B is a technology which enables production of a lot of recombinant proteins thanks to a low substrate delivering during 24 hours. This technology is based on polymer degradation by an enzyme leading to a control of the right quantity of substrate at each moment. We wanter to use this technology to cultivate our cells during one or two weeks in good conditions in order to produce butyrate and formate. The medium with the polymer was solid and contained in separate bags. To know which quantity of butyrate and formate we can produce, we had to know the quantity of substrate we could obtain with the polymer so we made a kinetic test with a high enzyme concentration (50 U/L).
Figure 21: Kinetic test of enzyme which degrades polymer from Biosilta kit. [Enzyme] = 50 U/L in order to have a complete degradation of polymer.
In order to have a global idea of the rate of glucose releasing we calculated an average speed.
$$ v_{glucose1}=\frac{[glucose]}{time}=\frac{11.1}{3.97}=2.80 g.L^{-1}.h^{-1} , for [E]_{1}=50 U.L^{-1} (4) $$
With a final glucose concentration of 13 g/L for one bag of polymer, a rate of glucose releasing can be calculated in order to have glucose during 13 days.
$$ v_{glucose2}=\frac{13}{13 days}=\frac{13}{322 hours}=0.0403 g.L^{-1}.h^{-1} (5) $$
The reduction factor was calculated:
$$ RF=\frac{v_{glucose1}}{v_{glucose2}}=\frac{2.8}{0.0403}=69.44 (6)$$
So, the concentration of enzyme that we had to use was:
$$ [E]_{2}=\frac{[E]_{1}}{RF}=\frac{50}{69.44}=0.72 U.L^{-1} (7)$$
Growth culture with Biosilta kit
As we do not know any growth characteristic with Biosilta medium we tested different enzyme concentrations and not only the one which allow growth during 13 days. We made acquisition in two times because of software constraints, this is why there is a break at 5 days:
Figure 22: Bacteria growth as a function of different enzyme concentrations in Biosilta medium. Test was made in 48 wells plate with OD reader.
Except for 1.5 U/L enzyme, OD increase during 12 days so glucose releasing seems to function well. At the beginning there is an exponential growth because some glucose is directly available on medium. Since 2 days until the end growth is linear, only the slope change. It is higher between 2 and 4 days than after probably because bacteria were in worse condition after few days so they were not able anymore to consume the remaining glucose.
Thanks to those results we know it is possible to have continuous growth during at least 12 days. The only problem is that our control, without enzyme, grows also so either another substrate is available or bacteria could able to degrade polymer that could be a problem.
In order to answer these questions we did culture in falcon in order to analyze products and to see evolution of polymer quantity. We made culture without enzyme and we test concentration of enzyme of 0.72 U/L because it this one which allows to reach the highest OD in figure 22 and it is the one we calculated above to have glucose releasing during 13 days.
Figure 23: Results of BW25113 culture on Biosilta medium without enzyme.
Figure 24: Results of BW25113 culture on Biosilta medium with [Enzyme] = 0.72 U/L.
Figure 23 shows that polymer is not degraded, so it is only enzyme of Biosilta kit which released glucose.
Enzyme concentration could be correlated to glucose releasing rate for further modeling. So,
bacteria found another carbon source in Biosilta medium but we were not able to determine which one.
Products concentrations are nearly identical to those in M9 medium.
In figure 24, polymer area decreased, so enzyme degraded it well.
At the beginning, glucose concentration was almost constant so bacteria consumed it directly when enzyme releases it. At the end glucose concentration increases a bit, bacteria either did not consume it as fast as the beginning or they consumed formate because its concentration decreases. This could be a problem for us because we look for produce more formate so we would have to think about it.
Fermentation products have high concentrations in comparison to culture in M9 with 15mM of glucose, around 20 times more for lactate, 3 times more for acetate and 2 times more for ethanol. So if we delete production ways for lactate, acetate and ethanol and degradation way of formate we would able to produce enough formate and butyrate.
Acids production modeling
With the rate of glucose calculated above, an FBA and FVA simulation were launched as explained in Modeling part.
Some conversion between the model and the real condition are necessary and they are also explained there.
In order to model production in the most similar conditions to fit real experiment we chose a glucose rate of 0.0403 g.L-1.h-1 that correspond to 0.72 U/L of enzyme.
To convert formate production into formic acid concentration we used equation (3).
Figure 25: Modeling of formic acid production as a function of different growth rates for a glucose rate of 0.0403 g.L-1.h-1.
Our goal was to produce at least 50µmol/L and with this graph the maximum production could be 6µmol/L. So we had to produce nearly 10 times more formic acid. In order to reach our goal we could see which rate of glucose we needed with a reverse thought.
Figure 26 : Modeling of formic acid production as a function of glucose rate for different growth rates (in h-1).
To produce 50 µmol/L of formic acid different strategies were available. Either a low growth rate could be chosen so that a low glucose rate would be necessary, or a high growth rate could be chosen and a high glucose rate would be necessary. As bacteria have to live during at least ten days it was better to have a continuous slow growth rate. Moreover, it would consume less glucose per hour so we would need a lower polymer concentration in our bag at the beginning. Thus, we chose a growth rate of 0.2h-1, and we could determinate the glucose rate needed.
[Formic acid] (μmol.L-1 )=166.88 ×[Glucose] (g.L-1.h-1 ) (9) $$ [Glucose] = \frac{50}{166.88}=0.3 g.L^{-1}.h^{-1} (10) $$
Now, we will see which butyric acid concentration we could have theorically produced.
Figure 27: Modeling of butyric acid production as a function of glucose rate for different growth rates.
According to modeling results in figure 10, we would have been able to produce around 100µmol/L of butyric acid
that corresponds to 0.0092% (V/V). As we explained in "Results" part, "attract" section,
our objective was to produce at least 0.00001%, so with modeling we reached it.
Nevertheless, in order to have this right glucose rate it was necessary to calculate how much polymer is needed at the beginning and which enzyme concentration.
With the same equations as we used in Characteristics of Biosilta kit
we could determine which quantity of glucose is needed in total during a fortnight.
$$[Glucose]=v_{glucose}\times time=0.3\times 322=96.6 g.L^{-1} (11)$$
Knowing that one Biosilta kit contains the equivalence of 13 g/L of glucose, we had to concentrate the medium 7 times. Concerning the glucose rate, the 0.3 g.L-1.h-1 value correspond to 5 U/L of enzyme. Thus, we tested different concentrations of Biosilta medium with different enzyme concentrations.
Testing different concentrations of Biosilta kit
As we did not know the exact composition of Biosilta medium, we are not able to say if there is a molecule which could be toxic at high concentrations. We could only have a global analysis on our results :
Figure 28: Bacteria growth as a function of time on Biosilta medium concentrated 6 times. Culture with BW25113 on 48 wells plate and optical reader
There was hardly any growth during three first days, bacteria probably adapted themselves to the medium. During 3 days until the end, OD increased up to 1 for 1.5 U/L enzyme but it was still slow. Moreover, bacteria grew better with 0.72 and 1.5 U/L than with 3 or 4 U/L. It could be explained by an excess of glucose that inhibits bacteria growth. Indeed, enzyme could release too much glucose, that bacteria would not consume this fast, then glucose accumulated itself in medium. We tested with a less concentrated medium in order to see if latency period could be reduce.
Figure 29: Bacteria growth as a function of time on Biosilta medium concentrated 4 times. Culture with BW25113 on 48 wells plate and optical reader
With a 4 times concentrated medium, there was no latency period anymore but enzyme concentration did not seem to affect bacteria growth. Bacteria probably consumed all free glucose in medium and then enzyme did not have enough time to degradate the polymer. A longer period test would have been necessary to know if bacteria were able to consume glucose as fast as the enzyme released it. Maybe by testing a twice concentrated medium, we would have been able to answer it.
Figure 30: Bacteria growth as a function of time on Biosilta medium concentrated twice. Culture with BW25113 on 48 wells plate and optical reader
Decline of curve for 0.72U/L was not expected because in “normal” Biosilta medium, figure 5, bacteria grew during 12 days. We cannot explain this result, but it shows that it is complicated to work with a medium with an unknown composition.
Curves for 3 and 4 U/L enzyme or very similar so it seems that bacteria are not able to consume all glucose released by enzyme. As we only measured OD we do not know if bacteria would assimilate glucose for another way that growth metabolism.
Thanks to figure 11, 12 and 13 we know that it would not be possible to have enough polymer in our medium. As a solution we think to use a dialysis system: in one side there will be bacteria and in the other side there will be the polymer with enzyme. Membrane which separates them will allow only small molecules to pass like glucose. Thanks to this system our device will have enough substrate for two weeks.
Regarding the rate of glucose assimilation we could do additional tests where we would measure glucose in medium to determine maximum rate of assimilation. An optimization of this assimilation could be essential.
Testing acids toxicity
Effects of medium
In order to optimize resistance of BW25113 to different acids concentrations we tested two medium: LB and M9 with 15mM of glucose.
Figure 31: Optic density in function of time for different formic acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
Figure 32: Optic density in function of time for different butyric acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
In M9 medium, growth was slower at the beginning in both figures but OD max was almost the same for both medium.
For formic acid, the only significant difference was for 10mM with a slower growth in LB than in M9. For butyric acid the difference was stronger because in LB bacteria did not grow anymore with 109mM, whereas in M9 there was growth.
In fact, M9 was buffered and not LB so we measured pH in both medium with different acids concentrations in order to see if there were a correlation.
Figure 33: pH in function of concentration in mM for formic acid and butyric acid. LB medium is represented with green curves and M9 medium with blue curves. pH was measured with pH paper because only order of magnitude interested us. Each condition was tested three times and give us the exactly the same results.
It is clear that in M9 medium pH stayed at pH 7 for higher acids concentrations than LB medium. Moreover, thanks to previous figures, it is possible to see that bacteria did not grow anymore when pH was around 5. This results show that bacteria are sensitive to acid pH, but they may have resisted to higher acids concentrations if the medium were better buffered. We will now see if it would be interesting or not to have buffered better our medium.
Formic acid toxicity
Figure 34: Toxicity test of formic acid, OD of BW on M9 15mM glucose.
Figure 34 shows a dose/response relationship between formic acid concentration and bacteria growth. We wanted to produce at least 50µmol/L of formic acid in order to kill varroas and bacteria growth normally up to 1mM. So, there should not have been toxicity problems during the treatment.
Butyric acid toxicity
Figure 35: Toxicity test of butyric acid, OD of BW on M9 15mM glucose.
The higher butyric concentration was, the less was bacteria growth, as our previous results with formic acid.
However we had an intermediate result with 109mM of butyric acid.
We did not have a specific butyric acid concentration to produce and modeling showed us that we could produce around 15mM with all optimizations,
so we would not have any butyric acid toxicity during our treatment.
Note: For our conclusions about acids toxicity, we consider that acids evaporate during day for formic acid and night for butyric acid,
so there would not be a lot of acid accumulation in the medium.
READ MORE
